Nitrosamines, determination

Reduction of a nitrosamine to a secondary amine. Proceed as for a nitro compound. Determine the solubility of the residue after evaporation of the ether and also its behaviour towards benzenesulphonyl (or p-toluenesulphonyl) chloride. 

Wet chemical methods determining titratable amine ate reported for products entering urethane (amine number as meq/g) or epoxy (AHEW = amine hydrogen equivalent weight) trade appHcations. For secondary amines /V-nitrosamine contaminants are reportable down to ppb using Thermoelectron Corporation thermal energy analy2er techniques. 

The iji vitro experiments, using the S-9 fraction from livers of uninduced Fisher 344 rats, was complicated by the fact that it became apparent that formaldehyde production was a poor measure of the extent of metabolism. The reason for that was that the S-9 fraction apparently catalyzed the oxidation of formaldehyde to formate. Consequently, determination of formaldehyde in an S-9 catalyzed reaction consistently gave low values of nitrosamine metabolism. Many workers use semicarbazide to suppress formaldehyde loss. We found, however, that semicarbazide is not a neutral bystander. 

The experiments with deuterium-labeled nitrosamines illustrate two important points. One is that oxidation of nitrosamines takes place at more than one position in the molecule, and the outcome of the balance of such competing reactions probably is the determinant of carcinogenic potency. The second is that the reason for the failure of carcinogenesis to be mirrored in many cases by the microsomally activated bacterial mutagenicity is that there can be several metabolic steps leading to formation of the proximate carcinogenic agent and not all of these need necessarily involve microsomal enzymes. ... 

A migration test was developed to simulate human exposure patterns. 5 g of rubber material was cut into 1-2 mm stripes and then immersed in 20 ml of standard test solution of artificial saliva (4.2 g NaHC03, 0.2 g K2CO3, 0.5 g NaCl, 0.03 g NaNO, ad 1000 ml with aqua dest.j. After 24 h incubation at 40 C volatile nitrosamines were determined in an aliquot and determined after destination with a standard technique (GC-TEA-method). 

The data in Table I are also significant in terms of the type of analysis to determine the presence of NDMA. In all cases analysis was done using gas chromatography coupled with a Thermal Energy Analyzer, a sensitive, relatively specific nitrosamine detector (12). Further, in six of the studies, the presence of NDMA in several samples was confirmed by gas chromatography-mass spectrometry (GC-MS). The mass spectral data firmly established the presence of NDMA in the beer samples. 

In Smoke. We compared the gas chromatograms of nitrosamines in matching aliquots of mainstream smoke derived from Burley type cigarettes that were identical except for the degree of nitrate fertilization during cultivation (Figure 2). This comparison supports the concept that the nitrate concentration in tobacco is a determining factor for the nitrosamine yields in the smoke. The data in Table II confirm this concept. These studies have... 

Nonvolatile Nitrosamines In Tobacco. A method which we developed several years ago for the analysis of tobacco-specific nitrosamines (TSNA 31) involves extraction of tobacco with buffered ascorbic acid TpH 4.5) followed by partition with ethyl acetate, chromatographic clean-up on silica gel, and analysis by HPLC-TEA (Figure 9). Results obtained with this method for a large spectrum of tobacco products (Table IV), strongly support the concept that the levels of nitrate and alkaloids, and especially the methods for curing and fermentation, determine the yields of TSNA in tobacco products. Recent and as yet preliminary data from snuff analyses indicate that aerobic bacteria play a role in the formation of TSNA during air curing and fermentation. 

We initiated an investigation to examine the possibility of N-nitrosamine formation from the herbicide glyphosate in different soils. It was also of interest to determine whether uptake of N-nitroso compound by plants would occur from soil. 

Reliable analytical methods are available for determination of many volatile nitrosamines at concentrations of 0.1 to 10 ppb in a variety of environmental and biological samples. Most methods employ distillation, extraction, an optional cleanup step, concentration, and final separation by gas chromatography (GC). Use of the highly specific Thermal Energy Analyzer (TEA) as a GC detector affords simplification of sample handling and cleanup without sacrifice of selectivity or sensitivity. Mass spectrometry (MS) is usually employed to confirm the identity of nitrosamines. Utilization of the mass spectrometer s capability to provide quantitative data affords additional confirmatory evidence and quantitative confirmation should be a required criterion of environmental sample analysis. Artifactual formation of nitrosamines continues to be a problem, especially at low levels (0.1 to 1 ppb), and precautions must be taken, such as addition of sulfamic acid or other nitrosation inhibitors. The efficacy of measures for prevention of artifactual nitrosamine formation should be evaluated in each type of sample examined. 

Reliable methods are available for determination of nitrosamines, especially volatile nitrosamines, in a variety of foods, environmental samples, commercial products, blood and animal tissues. Reviews of these methods are available (1, 2) and descriptions of some state-of-the-art procedures are included in papers on nitrosamine occurrence in this volume. This paper is not intended to be a comprehensive review of historical developments or of the many variations of procedures... 

An internal standard is desirable in any quantitative trace environmental analysis. The ideal internal standard should behave in a manner identical to that of the analyte in all the procedures followed for isolation, purification, and determination without producing interference. This is a difficult requirement to meet for nitrosamines, especially for NDMA. 

For the AOAC beer samples, a 2 m x 2 mm glass column packed with 8.57o Carbowax 20 M + 0.857, NaOH on 100/120 mesh Chromosorb G was used at 130 C and a helium flow rate of 20 cc/min. Retention times of NDMA and NDPA were 4.5 and 12.2 min, respectively. For the ASBC collaborative study, a 1 m x 2 mm glass column containing 67, Carbowax 20 M-TPA on 100/120 mesh Chromosorb G was operated at 90 C with 20 cc/min helium flow rate. Retention times were 3.6 and 11.3 min for NDMA and NDPA, respectively. For determination of nitrosamines in amines, a 2 m X 2 mm, 107, Carbowax 20 M-TPA on 100/120 mesh Chromosorb G column was operated at 190 C with a carrier gas flow rate of 20 cc/min. Retention times were NPYR, 6.6 min NMOR, 7.4 min. 

Morristown, NJ) for the ion source. No carrier gas separator was used. For determination of nitrosamines and TBDMS derivatives of hydroxy-nitrosamines, columns and operating conditions were identical to those for GC-TEA analyses For most work, the He flow rate was 15 cc/min and the column effluent was split 1 1 between a flame ionization detector and the mass spectrometer. The stainless steel splitter, solvent vent valve (Carle Instruments, Fullerton, CA), and associated plumbing were... 

It is clear that neither NMEA nor NDPA is appropriate for an internal standard in NDMA determination if criteria are interpreted strictly, but both compounds have been used for this purpose. Addition of a nitrosamine, not normally present in the sample, is helpful in detecting any gross errors in the procedure, but the addition should not be considered to be internal standardization. Utilization of NMEA or NDPA to indicate recovery of NDMA can lead to significant errors. In most reports of the application of these "internal standards", recovery of all nitrosamines was close to 100%. Under these conditions, any added compound would appear to be a good internal standard, but none is necessary. NDMA is a particularly difficult compound for use of internal standardization because of its anomalous distribution behavior. I mass j ectrometry is employed for quantitative determination, H- or N-labeled NDMA could be added as internal standard. Because the labeled material would coelute from GC columns with the unlabeled NDMA, this approach is unworkable when GC-TEA is employed or when high resolution MS selected ion monitoring is used with the equipment described above. 

Radioisotope-labeled nitrosamines have proven valuable in development of analytical methods and for demonstrating efficiency of recovery of nitrosamines from tobacco products and smoke (37-39). The very high specific activity required for low part-per-billion determinations has discouraged most analysts from using this approach. Unless a radiochromatographic detector with adequate sensitivity is available, samples must be counted independently of the final chromatographic determination, and one of the advantages of internal standardization, correction for variation in volume injected, is lost. 

Determination of Hydroxy-Nitrosamines. The column extraction procedure has proven flexible and convenient for isolating NDELA and BHP from a variety of matrices. No artifactual formation of these nitrosamines has been observed when sulfamic acid was incorporated with the sample. Addition of excess acid prevents elution of amines from the Celite column, minimizing nitrosation reactions at later stages. The triisopro-panolamine sample examined contained approximately 250 mg/kg BHP (Table IV). 

Direct Injection of Amines. In the course of developing methods for investigation of in vivo formation of NMOR and NPYR in rats treated with precursor amines and nitrite, it was necessary to determine the contamination levels of the amines by the nitrosamines. Spiegelhalder et al (32) reported the presence of nitrosamines in all secondary and tertiary amine samples which they examined, using vacuum steam distillation followed by extraction and GC-TEA determination. 

GCMS Analysis. High resolution MS may be used as the primary means for quantitative determination of nitrosamines (1, 11-13). Usually it is more convenient and efficient to use the TEA for this purpose and employ GCMS for confirmation of identity of nitrosamines tentatively identified by GC-TEA. 

This far into a nitrosamine symposium it should hardly be necessary to point out that nitrosamines are technically just one of a group of Ji-nitroso compounds that also includes nitros-amides, nitrosocarbamates, nitrosoureas, etc. Or that nitrosa-table pesticides encompass all the categories just mentioned and more. Or that many diverse pesticides, including herbicides, insecticides, and fungicides have been converted to Ji-nitroso derivatives in the laboratory (a recent review contained a 3-page, probably incomplete, compilation), or that some of the Ji-nitroso compounds thus synthesized were determined to be carcinogenic in test animals or mutagenic in various assays. 

Aromatic nitro and nitroso compounds are easily reduced at carbon and mercury electrodes. Other nitro compounds such as nitrate esters, nitramines, and nitrosamines are also typically easily reduced. The complete reduction of a nitro compound consists of three two-electron steps (nitro-nitroso-hydroxylamine-amine). Since most organic oxidations are only two-electron processes, higher sensitivity is typically found for nitro compounds. Several LCEC based determination of nitro compounds have been reported... 

---